Ovarian toxicity of 4-vinylcyclohexene diepoxide and polycyclic aromatic hydrocarbons in mice and rats

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are environmental ovotoxicants commonly released in cigarette smoke and vehicle exhaust. PAHs have been shown to target small ovarian follicles (25-100 μm) after a single high dose. Once irreplaceable primordial follicles are depleted in the ovary, ovarian failure results. The occupational chemical, 4-vinylcyclohexene diepoxide (VCD), induces pre-mature ovarian failure in mice and rats. A direct comparison between the ovotoxicity caused by VCD and that of PAHs was made by calculating an initial ovotoxic index (OI = ED₅₀ x 15d) in ovarian follicles of mice and rats. There are two hypotheses to be tested, first is that oocyte destruction can be prevented with a single dose of VCD. Second, three constituents of cigarette smoke, PAHs: 3-methylchloranthrene (3-MC), 9,10-dimethylbenzanthracene (DMBA) and benzo[a]pyrene (BaP) after repeated low-dose exposure results in apoptosis similar to VCD. VCD is protective against the normal rate of atresia in primary ovarian follicles following exposure to a single dose of VCD, and the mRNA expression of the cell death gene bax paralleled the changes in atresia. Using the initial OI, repeated low dose exposures were shown to be more toxic (lower OI) in the ovary than that previously reported from a single high dose. Furthermore, the calculation of the initial OI demonstrated DMBA to be the most potent chemical tested by destroying primordial follicles at the lowest dose. PAHs were also found to target secondary follicles. In the present study, the PAHs were also found to be associated with apoptosis (increased bax mRNA - DMBA and 3-MC; decreased bcl-x(L) mRNA - DMBA), in mice ovarian follicles. Confocal microscopy was used to visualize proteins of gene products from the bcl-2 family Bax (cell death) and Bcl-x(L) (cell survival) in mice. Large primary follicles illustrated consistently increased ratios of Bax/Bcl-x(L) for the three chemicals tested. These results suggest that repeated low dose exposure make ovarian follicles more susceptible to PAHs-induced cellular changes. Taken together with the confocal data, DMBA may be working through a different pathway than VCD and 3-MC, or the ovotoxic effects of DMBA are so potent that the effects due to 3-MC and VCD appear lower, and that large primary follicles may be the critical follicle stage determining the cells fate. The results obtained in these experiments can predict the potential reproductive effects initiated by VCD and PAHs.